Bottom Line:
Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively.Differentially expressed candidate genes were verified by RT-qPCR.We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro.

Background: Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC).

Methods: Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK.

Results: Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes.

Conclusion: We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

Fig3: Keratinocytes co-cultured with BLM cells exhibit accumulation of keratin 14-positive cells (A, K14, red signal) on the periphery of large colonies exhibiting keratins (green signal). This observation contrasts with the finding in co-cultures of keratinocytes with HPM cells (B). The measurements of the gradients of expression of K14 in both experimental situations (A1, B1) correlate with this observation. In case of keratinocytes co-cultured with BLM cells, this observation is further supported by the measurements of fluorescence intensity index (A2).

Mentions:
The results of co-culture with melanoma cells and NCSC cells were compared to the co-culture of keratinocytes with the HPM. In co-culture with BLM cells, HPK were strongly positive for K14 and for K8 (Figure 2A, B) and in lesser extent also for K19 (Figure 2C). BLM in this system also strongly increased the number of keratinocytes co-expressing keratins and vimentin together (Figure 2D). The BLM-conditioned medium had an inhibitory effect on proliferation of human keratinocytes as documented and quantified with advantage of feeder free growth of HaCaT (Additional file5: Figure S3). A very similar effect on HPK was also observed in co-culture with NCSC cells (Figure 2I-L). In the case of co-cultivation HPK with HPM, the expression of all studied markers was lower than in co-cultures with BLM and NCSC (Figure 2E-G).Interestingly, the expression of K14 was strong predominantly in small keratinocytes at the periphery of colonies far exceeding the intensity observed in the larger cells in the colony center (Figure 3).Figure 2

Fig3: Keratinocytes co-cultured with BLM cells exhibit accumulation of keratin 14-positive cells (A, K14, red signal) on the periphery of large colonies exhibiting keratins (green signal). This observation contrasts with the finding in co-cultures of keratinocytes with HPM cells (B). The measurements of the gradients of expression of K14 in both experimental situations (A1, B1) correlate with this observation. In case of keratinocytes co-cultured with BLM cells, this observation is further supported by the measurements of fluorescence intensity index (A2).

Mentions:
The results of co-culture with melanoma cells and NCSC cells were compared to the co-culture of keratinocytes with the HPM. In co-culture with BLM cells, HPK were strongly positive for K14 and for K8 (Figure 2A, B) and in lesser extent also for K19 (Figure 2C). BLM in this system also strongly increased the number of keratinocytes co-expressing keratins and vimentin together (Figure 2D). The BLM-conditioned medium had an inhibitory effect on proliferation of human keratinocytes as documented and quantified with advantage of feeder free growth of HaCaT (Additional file5: Figure S3). A very similar effect on HPK was also observed in co-culture with NCSC cells (Figure 2I-L). In the case of co-cultivation HPK with HPM, the expression of all studied markers was lower than in co-cultures with BLM and NCSC (Figure 2E-G).Interestingly, the expression of K14 was strong predominantly in small keratinocytes at the periphery of colonies far exceeding the intensity observed in the larger cells in the colony center (Figure 3).Figure 2

Bottom Line:
Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively.Differentially expressed candidate genes were verified by RT-qPCR.We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro.

Background: Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC).

Methods: Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK.

Results: Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes.

Conclusion: We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.